This application claims benefit of Japanese patent application JP 2000/152726 filed May 18, 2000.
The present invention relates to a paramyxovirus vector capable of transferring foreign genes and having a replication capacity.
Until now, most of the approaches of clinical studies pertaining to gene therapy have utilized viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses. These gene therapy vectors have limitations in gene transfer efficiency and continuous expression. Furthermore, the vectors themselves may have cytotoxicity, immunogenicity, and such problems are crucial when it comes to the medical application of these vectors (Lamb, R. A. and Kolakofsky, D., Paramyxoviridae: the viruses and their replication. in Fields Virology, 3rd edn, (Edited by B. N. Fields, D. M. Knipe and P. P. Howley) pp.1177-1204 (Philadelphia, Lippincott-Raven. (1996)). As a solution, novel vectors based on lentivirus and HSV have been proposed, and research is also being vigorously carried out to modify existing vectors. However, all these vectors exist in the form of DNA within the nucleus, throughout their life cycles. Therefore, it is difficult to overcome concerns of safety regarding random interactions with the chromosomes of the patient.
Rapid progress of reverse genetics technology is beginning to enable the development of vectors based on RNA viruses, which had long been delayed. Recombinant RNA vectors have a high gene transfer efficiency and expression capacity, and therefore, are highly potential vectors for gene therapy (Roberts, A. and Rose, J., K., Virology 247,1-6 (1998); Rose, J., Proc. Natl. Acad. Sci. USA 94, 14998-15000 (1996); Palese, P. et. al., Proc. Natl. Acad. Sci. USA 93, 11354-11358 (1996)). The paramyxovirus vectors that comprise negative strand RNA in the genome have several characteristics that significantly differ from retroviruses, DNA viruses, or plus strand RNA viruses. This genome or antigenome does not directly function as mRNA, and cannot initiate protein synthesis and genome replication of the virus. The RNA genome and antigenome of the virus consistently exists in the form of a ribonucleoprotein complex (RNP), and therefore problems of antisenses observed in plus strand RNA viruses, such as the inhibition of the assembly of genome towards RNP due to the hybridization of mRNAs to complementary naked genomic RNA, rarely occur. These viruses have their own RNA polymerase, and the transcription of viral mRNA or viral genome replication is carried out using the RNP complex as template. Worthy of special notice is the fact that negative strand RNA (nsRNA) viruses proliferate only within the cytoplasm of host cells, and since they do not have a DNA phase, they are not integrated into chromosomes. Furthermore, homologous recombination between RNAs has also not been observed. These features contribute largely to the stability and safety of negative strand RNA viruses as gene expression vectors.
Among the negative strand RNA viruses, the inventors have been focusing their attention on Sendai virus (SeV) that is not pathogenic towards humans, particularly the Z strain that is especially avirulent. SeV is a non-segmented negative strand RNA virus belonging to Paramyxoviruses, and is a type of murine parainfluenza virus. This virus attaches to the host-cell membrane via hemagglutinin-neuraminidase (HN) and fusion protein (F), which are two envelope glycoproteins, initiates membrane fusion, releases its own RNA polymerase and the RNA genome that exists in the form of ribonucleoprotein (RNP) complex into the cytoplasm, and carries out mRNA transcription and genome replication of the virus there (Bitzer, M. et al., J. Virol. 71(7): 5481-5486, 1997). The viral envelope protein F is synthesized as a non-active pre-protein (F0), is cleaved into F1 and F2 through proteolytic cleavage by trypsin (Kido, H. et al., Biopolymers (Peptide Science) 51(1): 79-86, 1999), and turns into an active protein causing membrane fusion. This virus is said to be non-pathogenic towards humans. Moreover, a laboratory-attenuated strain (Z strain) has also been isolated, which only induces a slight pneumonia in rodents, its natural hosts. This strain is widely used as a research model for molecular-level studies in the transcription/replication mechanisms of paramyxoviruses, and has also been used in the preparation of hybridomas. Apart from a high safety, this virus shows a high production titer of 109 to 1011 pfu/ml in cell lines and hen-eggs. In one recent successful recovery system from negative strand RNA virus cDNA, Sendai viruses showed an especially high reconstitution efficiency rate. In recombinant wild-type viruses transfected with foreign genes, the capacity to express the foreign gene efficiently as well stably, is gaining wide attention.
Even though Sendai viruses having a foreign gene upstream of the NP gene have been known, it was not known how the viral reconstitution and foreign gene expression will be affected when the foreign gene was inserted into a site other than the above.
An object of the present invention is to provide a paramyxovirus vector capable of transferring foreign genes and having a replicating capacity.
The inventors constructed viral vector DNA, in which a foreign gene was inserted to a site other than the upstream of the NP gene of the Sendai virus, and examined viral reconstitution and the expression level of the foreign gene. Namely, a new restriction enzyme site for inserting the foreign gene was introduced between the start signal and ATG translation start signal of each gene encoding viral proteins of Sendaivirus (SeV) full-length cDNA. A foreign gene (human secreted alkaline phosphatase (SEAP) gene) was inserted into this restriction enzyme site, and when reconstitution of the Sendai virus was conducted using LLC-MK2 cells, it was seen that only Sendai viruses having the capacity to proliferate were reconstituted. These viruses were amplified within hen-eggs to prepare a viral stock solution. These virus titers were combined and infected into LLC-MK2 cells, and the expression level of the foreign gene was determined to find that foreign gene expression is seen in all cases examined where the foreign gene was inserted into different sites. The foreign gene expression was relatively high when it was inserted between NP gene and P gene, or between P gene and M gene, and it was revealed that the foreign gene expression dropped as the site of insertion neared the downstream (5xe2x80x2 side of the negative strand) of the genome.
These results suggest that it is possible to obtain a relatively high foreign gene expression by placing the foreign gene downstream of the NP gene or the P gene, and that it is possible to decrease the expression level by placing the foreign gene towards the downstream of the genome. Based on these findings, it is possible to regulate the expression level of the foreign gene within the vector, by inserting the foreign gene into the upstream of the genome, namely, in the 3xe2x80x2 side of the negative strand genome, to obtain a high expression of the foreign gene. Conversely, when a high expression is not preferable, such as when the gene is cytotoxic, the foreign gene can be inserted downstream of the genome, namely, in the 5xe2x80x2 side of the negative strand genome. The paramyxovirus vector of the invention is useful in the expression of foreign genes in vivo and in vitro, and would especially be applied in gene therapy taking advantage of the outstanding features of paramyxoviruses.
Though problems regarding genomic stability may be pointed out in RNA viruses, results of heterologous gene expressions using SeV vector showed that there were hardly any nucleotide mutations even when the virus was serially passaged over several generations, and that it is possible to stably express the inserted heterologous genes over a long period of time (Yu, D. et al. Genes cells 2, 457-466 (1997)). Vectors based on this negative strand RNA viral replicons have several merits owing to their features, such as genomic stability, flexibility of packaging and size of the transfected gene due to not having capsid structure proteins, when compared to virus vectors based on replicons of already-successful positive strand RNA viruses, such as Semliki forest virus or Sindbis virus. At least 4 kbp of foreign DNA can be introduced into the replicable Sendai virus vector, and it may be also possible to simultaneously express more than two types of genes by adding a transcription unit. Vectors based on this Sendai virus replicon is expected to be continuously expressed since the replicated virus will re-infect surrounding cells, and the replicated RNP, multi copied in the cytoplasm of the infected cells, will be distributed to daughter cells following cell division. Moreover, the vector of the present invention could have an extremely broad tissue coverage and be high in application capacity since Sendai virus vectors are highly efficiently introduced into hematocytic cells, especially, granulocytic cells, and also to c-kit positive primitive cells.
Namely, the present invention relates to a paramyxovirus vector capable of transferring a foreign gene and having the capacity to replicate, and more specifically relates to,
(1) a replicable paramyxovirus vector carrying a foreign gene, wherein the foreign gene is located downstream of the genes encoding viral proteins in the negative strand genomic RNA contained within said vector,
(2) a replicable paramyxovirus vector carrying a foreign gene, wherein said vector is selected from the group consisting of the vectors of (a) to (f) below,
(a) a vector in which the foreign gene is inserted between the 1st gene encoding a viral protein and the 2nd gene encoding a viral structure protein from the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector,
(b) a vector in which the foreign gene is inserted between the 2nd gene encoding a viral protein and the 3rd gene encoding viral structure protein from the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector,
(c) a vector in which the foreign gene is inserted between the 3rd gene encoding a viral protein and the 4th gene encoding viral structure protein from the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector,
(d) a vector in which the foreign gene is inserted between the 4th gene encoding a viral protein and the 5th gene encoding viral structure protein from the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector,
(e) a vector in which the foreign gene is inserted between the 5th gene encoding a viral protein and the 6th gene encoding viral structure protein from the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector,
(f) a vector in which the foreign gene is inserted between the 6th gene encoding a viral protein from the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector, and the trailer sequence,
(3) the vector of (2), wherein the 1st to 6th genes encoding viral proteins from the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector are, NP gene, P gene, M gene, F gene, HN gene, and L gene, in their order,
(4) a DNA corresponding to the negative strand genomic RNA contained in the paramyxovirus vector of (1), or their complementary strands,
(5) a DNA corresponding to the negative strand genomic RNA contained in replicable paramyxovirus vector or its complementary strand, wherein said DNA comprises a cloning site for inserting a foreign gene downstream of the genes encoding viral proteins,
(6) a DNA corresponding to the negative strand genomic RNA contained in replicable paramyxovirus vector or its complementary strand, wherein said DNA is selected from the group consisting of the DNAs of (a) to (f) below,
(a) a DNA comprising a cloning site for inserting a foreign gene between the 1st and 2nd genes encoding viral proteins from the site equivalent to the 3xe2x80x2 end of the negative strand genomic RNA,
(b) a DNA comprising a cloning site for inserting a foreign gene between the 2nd and 3rd genes encoding viral proteins from the site equivalent to the 3xe2x80x2 end of the negative strand genomic RNA,
(c) a DNA comprising a cloning site for inserting a foreign gene between the 3rd and 4th genes encoding viral proteins from the site equivalent to the 3xe2x80x2 end of the negative strand genomic RNA,
(d) a DNA comprising a cloning site for inserting a foreign gene between the 4th and 5th genes encoding viral proteins from the site equivalent to the 3xe2x80x2 end of the negative strand genomic RNA,
(e) a DNA comprising a cloning site for inserting a foreign gene between the 5th and 6th genes encoding viral proteins from the site equivalent to the 3xe2x80x2 end of the negative strand genomic RNA,
(f) a DNA comprising a cloning site for inserting a foreign gene between the 6th gene coding a viral protein from the site equivalent to the 3xe2x80x2 end of the negative strand genomic RNA and the trailer sequence,
(7) the DNA of (6), wherein the 1st to 6th genes encoding viral proteins from the site equivalent to the 3xe2x80x2 end of the negative strand genomic RNA contained within the vector are, NP gene, P gene, M gene, F gene, HN gene, and L gene, in their order,
(8) a vector DNA carrying the DNA of (4) in an expressible manner, and,
(9) the vector DNA of (8), which carries positive strand genomic RNA in an expressible manner.
xe2x80x9cViral vectorxe2x80x9d as used herein indicates virions capable of transferring nucleic acid molecules into hosts. Paramyxoviruses in the present invention mean viruses or their derivatives belonging to the family Paramyxoviridae. Paramyxoviruses applicable in the present invention are, for example, parainfluenza virus type I including Sendai virus and human HA2, etc., parainfluenza virus type II including monkey SV5 and SV41, as well as human CA, etc., parainfluenza virus type III including bovine SF and human HA1, etc., parainfluenza virus type IV (including A subtype and B subtype), mumpus virus, Newcastle virus, and other various paramyxoviruses. The virus of the invention is more preferably a Sendai virus. These viruses may be natural strains, mutant strains, strains passaged in the laboratory, and man-made strains. Incomplete viruses, such as the DI particles (J. Virol. 68, 8413-8417 (1994)), etc., synthesized oligo nucleotides, and such may also be used as materials to prepare the viral vector of this invention.
Genes encoding the paramyxovirus protein include, NP, P, M, F, HN, and L genes. xe2x80x9cNP, P, M, F, HN, and L genexe2x80x9d refer to nucleocapsid, phospho, matrix, fusion, hemagglutinin-neuraminidase, and large protein encoding gene, respectively. The genes in each virus belonging to paramyxovirus sub-genus are generally indicated as below. Generally, NP gene may also be indicated as xe2x80x9cN gene.xe2x80x9d
For example, the accession numbers of the nucleotide sequence database of all genes of the Sendai virus classified into Respirovirus of Paramyxoviridae are, for the NP gene, M29343, M30202, M30203, M30204, M51331, M55565, M69046, X17218, for the P gene, M30202, M30203, M30204, M55565, M69046, X00583, X17007, X17008, for the M gene, D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, X53056, for the F gene, D00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152, X02131,for the HN gene, D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, X56131, for the L gene, D00053, M30202, M30203, M30204, M69040, X00587, X58886.
xe2x80x9cHaving a replication capacityxe2x80x9d or xe2x80x9cbeing replicablexe2x80x9d means that when the viral vector infects a host cell, the virus is replicated within said cell, and infective virions are produced. Also, in the present invention, xe2x80x9cDNAxe2x80x9d includes single-stranded and double-stranded DNA.